Mohler, Peter J.

Ankyrin B (AnkB) plays an important role in normal cardiac and neuronal function by linking membrane proteins to the cytoskeleton. Little is known, however, about the role of AnkB in the vasculature. Thus, we studied vasomotor function in aortas from wild‐type mice (AnkB +/+ ) and AnkB‐deficient littermates (AnkB +/− ). Maximum relaxation (M R ) to acetylcholine (Ach) was significantly reduced in AnkB +/− mice relative to AnkB +/+ mice (68 ± 5% vs 80 ± 1%), while responses to sodium nitroprusside were unaffected. While constrictor responses to PGF 2 α (1.5 ± 0.1g vs 1.4 ± 0.1g) were unchanged in AnkB +/− mice, constriction to phenylephrine was reduced by >35% in AnkB +/− mice compared to their AnkB +/+ littermates (p < 0.05). Incubation of AnkB +/+ vessels with the IP 3 ‐receptor inhibitor 2‐APB impaired M R to Ach in both AnkB +/+ and AnkB +/− mice (28 ± 6% and 29 ± 3%, respectively). Vasoconstrictor responses to phenylephrine were also dramatically attenuated by 2‐APB in AnkB +/+ mice (0.43 ± 0.05g vs 0.18 ± 0.02g) but not in AnkB +/− mice (0.28 ± 0.05g vs 0.27 ± 0.05g). These data suggest that Ankyrin B plays a significant role in determining both endothelial and vascular smooth muscle function, in part through the modulation of the IP 3 receptor. Future studies examining the molecular mechanisms whereby Ankyrin B affects vasomotor function may lend critical insights into vasomotor dysfunction in health and disease.

The gene encoding SAP97 ( DLG1) joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1-encoded SAP97’s candidacy as a minor Brugada syndrome susceptibility gene.

Objective This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). Design All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. Results The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A , the gene that encodes the Na + channel Na V 1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na + current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. Conclusions A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na + channel Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

AbstractBackgroundThe SCN5A‐encoded voltage‐gated sodium channel NaV1.5 is expressed in human jejunum and colon. Mutations in NaV1.5 are associated with gastrointestinal motility disorders. The rat gastrointestinal tract expresses voltage‐gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown.MethodsThe presence and distribution of Scn5a‐encoded NaV1.5 was examined by PCR, Western blotting and immunohistochemistry in rat jejunum. Freshly dissociated smooth muscle cells were examined by whole cell electrophysiology. Zinc finger nuclease was used to target Scn5a in rats. Lentiviral‐mediated transduction with shRNA was used to target Scn5a in rat jejunum smooth muscle organotypic cultures. Organotypic cultures were examined by sharp electrode electrophysiology and RT‐PCR.Key ResultsWe found NaV1.5 in rat jejunum and colon smooth muscle by Western blot. Immunohistochemistry using two other antibodies of different portions of NaV1.5 revealed the presence of the ion channel in rat jejunum. Whole cell voltage‐clamp in dissociated smooth muscle cells from rat jejunum showed fast activating and inactivating voltage‐dependent inward current that was eliminated by Na+ replacement by NMDG+. Constitutive rat Scn5a knockout resulted in death in utero. NaV1.5 shRNA delivered by lentivirus into rat jejunum smooth muscle organotypic culture resulted in 57% loss of Scn5a mRNA and several significant changes in slow waves, namely 40% decrease in peak amplitude, 30% decrease in half‐width, and 7 mV hyperpolarization of the membrane potential at peak amplitude.Conclusions & InferencesScn5a‐encoded NaV1.5 is expressed in rat gastrointestinal smooth muscle and it contributes to smooth muscle electrophysiology.

Human jejunum smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs) express the SCN5A-encoded voltage-gated, mechanosensitive sodium channel NaV1.5. NaV1.5 contributes to small bowel excitability, and NaV1.5 inhibitor ranolazine produces constipation by an unknown mechanism. We aimed to determine the presence and molecular identity of Na+ current in the human colon smooth muscle and to examine the effects of ranolazine on Na+ current, mechanosensitivity, and smooth muscle contractility. Inward currents were recorded by whole cell voltage clamp from freshly dissociated human colon SMCs at rest and with shear stress. SCN5A mRNA and NaV1.5 protein were examined by RT-PCR and Western blots, respectively. Ascending human colon strip contractility was examined in a muscle bath preparation. SCN5A mRNA and NaV1.5 protein were identified in human colon circular muscle. Freshly dissociated human colon SMCs had Na+ currents (−1.36 ± 0.36 pA/pF), shear stress increased Na+ peaks by 17.8 ± 1.8% and accelerated the time to peak activation by 0.7 ± 0.3 ms. Ranolazine (50 μM) blocked peak Na+ current by 43.2 ± 9.3% and inhibited shear sensitivity by 25.2 ± 3.2%. In human ascending colon strips, ranolazine decreased resting tension (31%), reduced the frequency of spontaneous events (68%), and decreased the response to smooth muscle electrical field stimulation (61%). In conclusion, SCN5A-encoded NaV1.5 is found in human colonic circular smooth muscle. Ranolazine blocks both peak amplitude and mechanosensitivity of Na+ current in human colon SMCs and decreases contractility of human colon muscle strips. Our data provide a likely mechanistic explanation for constipation induced by ranolazine.

Abstract:Over the past 15 years, gene mutations in cardiac ion channels have been linked to a host of potentially fatal human arrhythmias including long QT syndrome, short QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia. More recently, a new paradigm for human arrhythmia has emerged based on gene mutations that affect the activity of cardiac ion channel- and transporter- associated proteins. As part of theCirculation Researchthematic series on inherited arrhythmias, this review focuses on the emerging field of human arrhythmias caused by dysfunction in cytosolic gene products (including ankyrins, yotiao, syntrophin, and caveolin-3) that regulate the activities of key membrane ion channels and transporters.

The National Heart, Lung, and Blood Institute and Office of Rare Diseases at the National Institutes of Health organized a workshop (September 14 to 15, 2006, in Bethesda, Md) to advise on new research directions needed for improved identification and treatment of rare inherited arrhythmias. These included the following: (1) Na + channelopathies; (2) arrhythmias due to K + channel mutations; and (3) arrhythmias due to other inherited arrhythmogenic mechanisms. Another major goal was to provide recommendations to support, enable, or facilitate research to improve future diagnosis and management of inherited arrhythmias. Classifications of electric heart diseases have proved to be exceedingly complex and in many respects contradictory. A new contemporary and rigorous classification of arrhythmogenic cardiomyopathies is proposed. This consensus report provides an important framework and overview to this increasingly heterogeneous group of primary cardiac membrane channel diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular biology and novel therapeutic approaches in cardiology, as well as the introduction of many recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy in consensus with a recent American Heart Association Scientific Statement.